Staphylococcus aureus is a major cause of community and hospital-acquired infections of the skin, soft tissue, and bloodstream. The recent dramatic increase in occurrence of strains resistant to beta-lactam antibiotics (MRSA) has reduced therapeutic options significantly, but most MRSA strains remain sensitive to other bactericidal antibiotics in the fluoroquinolone (FQ) and glycopeptide (GP) classes. Recent research has demonstrated that bactericidal antibiotics stimulate the production of reactive oxygen species (ROS) in bacteria, which contribute to cell death in a manner similar to the host immune oxidative burst. Alleviation of this oxidative stress by the production of nitric oxide (NO) by bacterial NO synthase (bNOS) enhances the survival of several bacterial species, including staphylococci, to antibacterial therapy and to neutrophil killing. Consequently, small molecule bNOS inhibitors will provide an adjunctive therapeutic approach to bolster the effectiveness of antibiotics and neutrophils against MRSA and potentially reduce the selective pressure for development of drug-resistance by increasing the duration of effective circulating levels of antibiotic. Substantial differences between mammalian and bacterial NOS enzymes indicate that selective inhibition of bNOS is feasible. The overall goal of this project is to discover and develop drugs that increase the efficacy of clinically relevant bactericidal antibiotics against pathogenic staphylococci such as MRSA by specifically inhibiting bacterial NO production. Our strategy is to identify small molecule bNOS inhibitors and to develop them into innovative adjunctive therapies that increase the bactericidal activity of FQ, GP, and beta-lactam antibiotics. In preliminary studies, we established proof of concept for bNOS as a novel target for adjunctive therapy by demonstrating that the growth and viability of strains of S. aureus, B. subtilis, and B. anthracis carrying deletions of the bNOS gene were more sensitive to several bactericidal antibiotics than were their wild-type parents. NO in bacterial cells was shown to activate catalase, induce sodA (superoxide dismutase), suppress the Fenton reaction (production of reactive hydroxyl radicals from H2O2), and rescue cells from ROS generated by bactericidal drugs and by the immune oxidative burst. These results indicate that bactericidal drugs can be potentiated by targeting bacterial systems that reduce ROS damage, such as bNOS. In Phase I, we will construct and optimize luminescent, fluorescent, and biochemical primary and secondary screening assays for bNOS inhibitors, apply them to libraries representing >300,000 discrete chemical compounds, confirm the hits, and validate them as potent, selective potentiators of several antibiotics and neutrophils vs. MRSA and other drug- resistant species. The most potent, broadest acting bNOS inhibitors will be characterized to eliminate compounds with off-target activity vs. the 3 mammalian NOS isozymes and cytotoxicity. In Phase II, we will develop the most promising validated hits into lead compounds by optimizing their activity and specificity using rational drug design and evaluate them for efficacy and toxicity in animal models of infection.